Glaucoma is a major cause of blindness in the world and is characterized by a loss of retinal ganglion cells. The disease is frequently associated with elevated intraocular pressure, but it is unclear how this elevation leads to the death of ganglion cells. The current proposal is based upon the novel hypothesis that elevated intraocular pressure triggers the release of ATP. This released ATP can activate P2X7 ATP receptors on retinal ganglion cells, leading to activation of NMDA receptors and excitotoxic cell death. Alternatively, the released ATP can be converted into adenosine by ecto-enzymes and stimulate the A3 receptor for adenosine and protect ganglion cells. Three approaches will be used to test this hypothesis. First, the neuroprotective capacity of rat A3 receptors will be investigated pharmacologically, as this represents the most straightforward opportunity for treatment. Receptor identity will be confirmed by amplifying mRNA message from fluorescently-labeled retinal ganglion cells isolated using Laser-Capture Microdissection. Secondly, the interaction between P2X7 and NMDA receptor channels will be explored by examining the ability of NMDA antagonists to prevent the cell death caused by P2X7 stimulation. The mechanism underlying this interaction will be examined by measuring the effect of P2X7 stimulation on glutamate efflux, and determining the permeability of the P2X7 channel itself to glutamate. Indirect pathways for interaction will also be explored, investigating whether P2X7 receptor stimulation depolarizes cells sufficiently to open NMDA channels or trigger a vesicular release of glutamate. Finally, the effect of increased pressure on ATP release will be tested using in vitro and in vivo models. The ability of elevated pressure to trigger ATP release from dissociated rat retinal cells will be confirmed, and the effect of changing the magnitude and duration of this pressure will be investigated. The hypothesis will be tested with a rat model of experimental glaucoma by correlating the magnitude and duration of intraocular pressure elevation with levels of ATP and regulating enzymes. Together this research will provide a new explanation for ganglion cell death in glaucoma and suggests several new approaches to prevent this death.